The migration of vascular smooth muscle cells (VSMCs) from the media to the intima and the proliferation of intimal VSMCs are key events in restenotic lesion development. These events, which are preceded and accompanied by inflammation, are modulated by the proinflammatory cytokine, interleukin-1 beta (IL-1 beta), which induces vascular smooth muscle cells to express adhesion molecules and to proliferate. IL-1 beta action is complex and regulated, in part, by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1ra). Whether there was a temporal and spatial correlation between IL-1 beta and IL-1ra expression in, and release by, oxidized low density lipoproteins (oxLDL)-stimulated human aortic smooth muscle cells (HASMCs) was determined by using ELISA and Western blot. In addition, IL-1 beta and IL-1ra expression was detected in the neointima of endothelia-denuded cholesterol-fed New Zealand white rabbits by immunohistochemistry and Western blot. In HASMCs, oxLDL induced IL-beta and IL-1ra expression and release in a dose- and time-dependent manner. Treatment with 20 microg/ml oxLDL resulted in increased IL-1 beta release after 6 h, which peaked at 24 h, and in increased IL-1ra release, first seen after 12 h, but continuing to increase for at least 48 h. In the cells, IL-beta expression showed a similar pattern to release, whereas IL-1ra expression was seen in unstimulated cells and was not increased by oxLDL treatment. Confocal microscopy showed colocalization of IL-beta and IL-1ra expression in oxLDL-stimulated HASMCs. oxLDL caused significant induction of nuclear factor kappa B and activator protein-1 DNA binding activity in HASMCs (6.6- and 3.3-fold, respectively). In cholesterol-fed endothelia-denuded rabbits, the notably thickened intima showed significant IL-1 beta and IL-1ra expression. These results provide further support for the role of IL-1 system in the pathogenesis of restenosis. This is the first demonstration of IL-1 beta and IL-1ra expression and secretion of oxLDL-treated HASMCs and their expression in the rabbit neointima, suggesting that the smooth muscle cells of the intima are an important source of these factors.
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"g/ml) was isolated by sequential ultracentrifugation of fasting plasma samples from healthy adult males and extensively dialyzed under nitrogen for 24 h at 4 °C against phosphate-buffered saline (PBS, 5 mmol/l phosphate buffer and 125 mmol/l NaCl, pH 7.4). This native LDL (nLDL) was oxidized by dialysis for 24 h at 37 °C against 10 µmol/l CuSO 4 in PBS, as previously described . The extent of oxidation was monitored by measuring thiobarbituric acid-reactive substance (TBARS) and agarose gel electrophoresis. "
[Show abstract][Hide abstract]ABSTRACT: In a variety of vascular disorders, endothelial cells (ECs) are exposed to high levels of reactive oxygen species (ROS) generated intercellularly. Recently, several anti-oxidants, including catalase, have been suggested to be cytoprotective against the development of atherosclerosis. The object of this study was to investigate whether adenovirus-mediated gene transfer of catalase in ECs can attenuate ROS production and cell apoptosis under oxidized low density lipoprotein (oxLDL) stimulation. Adenovirus-mediated gene transfer of human catalase gene (Ad-Cat) resulted in a high level of catalase overexpression in human arterial EC (HAEC), which manifested a time-dependent increase in cell viability under the exposure of oxLDL and decreased oxLDL-induced apoptosis. Phosphorylation studies of ERK1/2, JNK, and p38, three subgroups of mitogen activator protein kinase demonstrated that catalase overexpression suppressed JNK phosphorylation and increased ERK1/2 phosphorylation. NF-kappaB and AP-1 were induced after the exposure of HAECs to oxLDL. While catalase overexpression was found to inactivate AP-1, it had no effect on NF-kappaB activity. These results provide the evidence that overexpression of catalase in ECs attenuates ROS production and cell apoptosis under oxLDL stimulation. The protective effect is mediated through the downregulation of JNK and the upregulation of ERK1/2 phosphorylation as well as AP-1 inactivation. This observation supports the feasibility of catalase gene transfer to human endothelium to protect against oxidant injury.
Full-text · Article · Feb 2004 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract]ABSTRACT: Low density lipoproteins (LDLs) modulate the expression of key genes involved in atherogenesis. Recently, we have shown that the transcription factor neuron-derived orphan receptor-1 (NOR-1) is involved in vascular smooth muscle cell (VSMC) proliferation. Our aim was to analyze whether NOR-1 is involved in LDL-induced mitogenic effects in VSMC.
LDL induced NOR-1 expression in a time- and dose-dependent manner. Antisense oligonucleotides against NOR-1 inhibit DNA synthesis induced by LDL in VSMCs as efficiently as antisense against the protooncogene c-fos. The upregulation of NOR-1 mRNA levels by LDL involves pertusis-sensitive G protein-coupled receptors, Ca2+ mobilization, protein kinases A (PKA) and C (PKC) activation, and mitogen-activated protein kinase pathways (MAPK) (p44/p42 and p38). LDL promotes cAMP response element binding protein (CREB) activation (phosphorylation in Ser133). In transfection assays a dominant-negative of CREB inhibits NOR-1 promoter activity, while mutation of specific (cAMP response element) CRE sites in the NOR-1 promoter abolishes LDL-induced NOR-1 promoter activity.
In VSMCs, LDL-induced mitogenesis involves NOR-1 upregulation through a CREB-dependent mechanism. CREB could play a role in the modulation by LDL of key genes (containing CRE sites) involved in atherogenesis.
Preview · Article · May 2004 · Arteriosclerosis Thrombosis and Vascular Biology